All orchids maintain an obligate relationship with mycorrhizal symbionts during seed germination. In most cases, germination-enhancing fungi have been isolated from roots of mature plants for conservation and cultivation purposes. To understand the germination biology of Dendrobium devonianum, an over-collected medicinal orchid, the seeds of D. devonianum were inoculated with a fungal strain (FDd1) isolated from naturally occurring protocorms of D. devonianum and two other germination-enhancing fungal strains (FDaI7 and FCb4) from D. aphyllum and Cymbidium mannii, respectively. The fungal strain was isolated from five protocorms of D. devonianum and identified as a species of the genus Epulorhiza. In germination trials, treatments with all of the three fungal strains showed a significant promoting effect on seed germination and protocorm formation, compared with the control treatment (no inoculation). However, FDd1 fungal strain showed the greatest effectiveness followed by FDaI7 and FCb4. For all inoculation and control treatments, seeds developed to protocorms regardless of the presence of illumination, whereas protocorms did not develop to seedlings unless illumination was provided. The results of our manipulative experiments confirmed the hypothesis that mycorrhizae associated with orchid seedlings are highly host-specific, and the degree of specificity may be life stagespecific under in vitro conditions. The specific mycorrhizal symbionts from protocorms can enhance restoration efforts and the conservation of orchids such as D. devonianum.
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Based on this background, there were three primary goals of this study. Firstly, to investigate the key anatomical features during zygotic embryo development of P. armeniacum in association with the ability of zygotic embryos to germinate asymbiotically in vitro. Secondly, to examine the effect of culture media and seed pretreatments on seed germination and subsequent seedling growth, with the ultimate purpose of increasing both. Thirdly, to establish an effective in vitro propagation system for the large-scale propagation of P. armeniacum to meet commercial needs and to eventually reestablish populations of this threatened orchid species back into the wild.
At 180 days after culture, 95 DAP seeds showed highest germination percentage (96.2%), which was significantly higher than all other collection periods, but the percentage of protocorm necrosis was also highest (20.0%). Although the next highest seed germination percentage was 75.0% in 110 DAP seeds, a value that was also significantly higher than all other collection periods, 50% of the protocorms developed to stage 5. This value was significantly higher than all other collection periods (Table 3). Therefore, 110 DAP seeds were assumed to be most suitable for protocorm development.
Seeds of 95 or 110 DAP showed a different response to most basal media at 180 days after culture (Table 4 and Table 5). Highest total seed germination percentage of 95 DAP seeds was observed on quarter-strength Murashige and Skoog23 (MS) and on eighth-strength (MS) basal media relative to the other eight tested basal media. However, MS was most suitable for subsequent protocorm development. When MS was used, seed germination percentage at stage 5 was 40.5%, which was significantly higher than the other nine tested basal media, including 30.1% seed germination on MS medium. This latter medium was most suitable for germination of 110 DAP seeds and subsequent protocorm development, resulting in the highest total seed germination percentage (75.0%) with 50% of protocorms developing to stage 5. These values were significantly higher than on the other nine tested basal media. Therefore, MS medium proved to be the most appropriate basal medium for seed germination and protocorm development of 95 DAP seeds while MS was most suitable for 110 DAP seeds.
Plantlets grew vigorously 30 days after transplanting. After 90 days of transplanting, the highest percentage of plantlet survival (90.7%) was observed on Chilean sphagnum moss which was significantly higher than on sieved peat, commercial sand for orchids or substrate mixture 2 (Table 9). The lowest plantlet survival percentage (74.3%) was observed on commercial sand for orchids, which was not significantly different to sieved peat or substrate mixture 2. After 180 days of transplanting, the highest plantlet survival percentage (85.3%) was observed on Zhijing stone for orchids (Fig. 3j), which was also significantly higher than on sieved peat, commercial sand for orchids or substrate mixture 2, but the lowest plantlet survival percentage (63.7%) was observed on commercial sand for orchids. This latter substrate performed most poorly among all tested substrates. Relative to plantlet survival percentage on the same substrate at 90 and 180 days after transplanting, both values were not significantly different on Zhijing stone for orchids, substrate mixture 1 or substrate mixture 3; all three substrates included Zhijing stone for orchids (Table 9). Since the roots of transplanted seedlings almost never elongated on Chilean sphagnum moss, Zhijing stone for orchids is most suitable for transplanting seedlings. About 5000 plantlets from all treatments were successfully acclimatized to greenhouse conditions and can be used for ornamental, ecorehabilitation and conservation purposes.
Microscopy showed that the roots of analysed C. inverta samples were extensively colonized by fungal hyphae forming pelotons in root cortical cells. Fungal ITS regions were amplified by polymerase chain reaction, from DNA extracted from fungal mycelia isolated from orchid root samples, as well as from total root DNA. Molecular sequencing and phylogenetic analyses showed that the investigated orchid primarily associated with ectomycorrhizal fungi belonging to a narrow clade within the family Ceratobasidiaceae, which was previously detected in a few fully mycoheterotrophic orchids and was also found to show ectomycorrhizal capability on trees and shrubs. Russulaceae fungal symbionts, showing high similarity with members of the ectomycorrhizal genus Russula, were also identified from the roots of C. inverta, at young seedling stage. Ascomycetous fungi including Chaetomium, Diaporthe, Leptodontidium, and Phomopsis genera, and zygomycetes in the genus Mortierella were obtained from orchid root isolated strains with unclear functional role.
Chamaegastrodia inverta adult and completely developed plants (a, b, c) and young hypogeous individuals (seedlings, d). Green leaves and stems represented in Fig. 1 a belong to non-orchid surrounding vegetation in C. inverta habitat
Total root DNA sequencing revealed that mycorrhizal tissue was dominated by fungi belonging to the family Ceratobasidiaceae (Table 1). For 7 out of the total 8 investigated C. inverta plants, amplified sequences were related to Ceratobasidium sequences in GenBank. The young orchid plant (seedling) sample 6 yielded sequences 6a (accession no. MT278316) and 6b (MT278317) with close identity to GenBank accession sequences of Russulaceae and Ceratobasidiaceae, respectively (Table 1).
The use of different experimental approaches, including morphological analysis combined with molecular sequencing, following both culture-dependent methods and direct total orchid root DNA amplification, allowed the detection and identification of C. inverta root-associated fungi, in different plant life stages. Results showed that the primary mycorrhizal symbionts of C. inverta are within the family Ceratobasidiaceae. This family is included in the diverse group of Rhizoctonia-like fungi, which comprises a range of rather distantly related fungal taxa characterised by homogenous asexual stage hyphal morphology (90 branching of hyphae, a constriction at the branch point, and a septum near the point of origin in the branch hyphae), as well as by a common significant predisposition to establish mycorrhizal symbiosis with orchids [4, 31, 32]. The morphology of mycelia forming pelotons in the root cells of analysed C. inverta individuals, showing typical Rhizoctonia features, is consistent with molecular identification of Ceratobasidiaceae fungal symbionts. Ceratobasidioid fungi have been previously found to associate with several other orchids including both tropical and temperate species [32,33,34]. Members of this fungal family have been recognized as important associates in epiphytic orchids belonging to different genera, such as Oncidium [35], Ionopsis and Tolumnia [36, 37], as well as in terrestrial orchids, of both forest and meadow habitats, including Goodyera [38,39,40], Anacamptis, Cephalanthera and Orchis [16, 34, 41]. Although mycorrhizal associations with Ceratobasidiaceae involve orchids with very different biogeographical and ecological features, the great majority of orchid species that have been found to establish a trophic relationship with Ceratobasidiaceae fungi belong to the same physiological category of green orchids, including species with different degrees of photosynthetic capability, from fully autotrophic to mixotrophic species [8, 42, 43]. Achlorophyllous non-photosynthetic orchids, instead, are almost completely excluded from mycorrhizal partnerships with Ceratobasidiaceae, with very few exceptions [28, 44]. Our finding of ceratobasidioid fungi as dominant associates in the roots of the achlorophyllous forest orchid C. inverta represents a new record of the rare association between the identified fungal group and fully mycoheterotrophic orchids in nature. Phylogenetic relationships reconstructed from rDNA sequence information suggest that the C. inverta associated Ceratobasidiaceae have limited genetic diversity and likely belong to the same species (Fig. 3). Chamaegastrodia inverta mycobionts are phylogenetically close to a peculiar group of Ceratobasidiaceae showing ectomycorrhizal capability, such as the fungi previously found in C. shikokiana in Japan, which were also able to form ectomycorrhizas on the rootlets of the woody plant species Abies firma sedlings in pot culture [28], and the fungi associated with the Australian subterranean orchid (flowering below ground) Rhizanthella gardneri [44]. Mycorrhizal fungi, isolated from pelotons extracted from the rhizomes of the latter orchid species, were tested by Bougoure and collaborators for their ability to form ectomycorrhizal associations with several plant species belonging to the genus Melaleuca, which resulted in the undoubted formation of mantle and Hartig net, typical ectomycorrhizal structures [44]. Ectomycorrhizal taxa constitute a rare exception among Ceratobasidiaceae, the great majority of members within this fungal family, as well as Rhizoctonia-forming Agaricomycotina in general, being regarded as saprotrophs and plant pathogens [28, 31, 43,44,45]. The previously reported cases of tripartite relatioships C. shikokiana-Ceratobasidium-A. firma [28] and R. gardneri-Ceratobasidium-Melaleuca [44] involved ceratobasidioid fungi which were able to establish orchid mycorrhizas with fully mycoheterotrophic orchid species and ectomycorrhizas with autotrophic tree or shrub hosts simultaneously, the photosynthetic plant partner being the provider of carbon for the system. An additional example of ectomycorrhizal Ceratobasidiaceae was provided by the fungi isolated from the roots of the mixotrophic orchid Platanthera minor, which were also found to show ectomycorrhiza-forming ability on Pinus densiflora, the sole ectomycorrhizal tree species in the sampling sites in Japan [45]. The P. minor associated Ceratobasidium fungi formed a highly supported clade with mycobionts from C. shikokiana and R. gardneri in the phylogenetic analyses performed by Yagame et al. [45]. The Ceratobasidiaceae associated with C. inverta, in this study, are also closely related and cluster with the above mentioned ectomycorrhizal ceratobasidioid fungi from C. shikokiana and R. gardneri. It is therefore possible that C. inverta establishes a tripartite relationship with some of the surrounding autotrophic plants in the forest habitats where it grows, using the ceratobasidioid associated fungi to exploit the ectomycorrhizal plant as a carbon source. Further studies are necessary to test this hypothesis, by determining C. inverta natural abundance in 13C and 15N compared with those of surrounding photosynthetic plants, in order to confirm and quantify the contribution of fungal source to the orchid carbon nutrition [46]. Besides, experiments of ectomycorrhiza formation on roots of potential tree hosts available in C. inverta habitats, using Ceratobasidiaceae isolated from the studied orchid as inoculum, would clarify the ability of the orchid fungal associates to establish ectomycorrhizal symbiosis with surrounding autotrophic partners. Our attempt of fungal isolation from pelotons extracted from the cortical cells of C. inverta roots was unsuccessful. The sole strain observed to apparently grow from a peloton isolated from orchid sample 5 was molecularly similar to an uncultured fungus detected in agricultural soil from Japan [47] and to a Mortierella sequence amplified from Zea mays field soil samples in Germany [48] (Table 2). This isolated mycelium may actually reflect a fungal contaminant from spore or a non-mycorrhizal root endophyte from hyphal fragment accidentally trapped in the cultured peloton. The difficulty in isolating in axenic culture the real C. inverta fungal symbionts from pelotons was confirmed by the result of isolation attempts from root fragments, which provided a number of ascomycetous and zygomycetous strains, but did not yield any ceratobasidioid basidiomycete. This result is in agreement with previous studies on C. shikokiana and R. gardneri, involving similar Ceratobasidiaceae mycorrhizal fungi. In the former study on C. shikokiana the authors reported that no fungal growth was observed on Czapek-Dox medium, which they normally used for saprobic fungi isolation, while active mycelial growth was obtained from pelotons cultured on a Modified Melin-Norkrans medium, specific for ectomycorrhizal fungi [28]. Similarly, in the study on R. gardneri, Bougoure et al. [44] found that isolation and growth of fungal pelotons from the roots of the studied orchid were mostly unsuccessful, with extracted hyphal coils failing to grow or colonies suddenly dying after initial growth. The absence of hyphal growth from C. inverta mycobiont extracted pelotons on PDA medium suggests that the analysed fungi belong to the ectomycorrhizal trophic group and may require very specific media to be isolated. However, the ecology and lifestyle of Ceratobasidiaceae associated with achlorophyllous mycoheterotrophic orchids is difficult to predict and generalize, and requires in-depth analyses to be understood, in every single association with different plant species. For instance, Bougoure et al. [49] showed that Ceratobasidiaceae associated with R. gardneri obtained carbon by both saprothrophic and mycorrhizal means, simultaneously. In the latter work, isotopically labelled tracers, 13CO2 and double-labelled [13C-15N]glycine were used to assess the direction of carbon and nitrogen transfers between the plants involved in the investigated tripartite association via the fungal connections, showing that R. gardneri obtained nutrients from the associated mycorrhizal ceratobasidioid fungi, which were able to derive carbon not only from surrounding autotrophic shrubs via ectomycorrhizas, but also from soil organic matter via saprotrophic activity [49]. 2ff7e9595c
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